Inwardly rectifying K (Kir) channels set the resting membrane potential of a cell. They also help the generation of action potential by carrying less current at the depolarized potentials. Neurotransmitters or intracellular ATP can regulate some Kir channels to adjust cell function. The molecular properties, structure, ion selectivity, rectification, and gating of Kir channels have been studied extensively. In contrast, relatively little attention has been paid to the interaction of these channels with their surrounding lipid environment. Previously, these investigators found that an interaction between the charged headgroups of phospholipids and a C-terminal region of Kir channels regulates channel activation. In addition, it has been realized previously that lipid metabolites such as lysopholipids can affect Kir channel current. The long-term goal of this project is to understand how the lipid/channel interaction changes the gating of Kir channels. A typical Kir channel is a tertramer of four subunits, each containing two transmembrane helices. This "thin wall" structure allows a possible interface between membrane lipids and channel domains in control of gating. The current working hypothesis is described by a dual-interaction model. First, a specific interaction between the lipid headgroup and the cytoplasmic domain of the Kir channel causes the channel to open. Second, through a nonspecific interaction, the lipid hydrocarbon chain restricts the gating movement of M2 helices. To test this hypothesis, the investigators propose to study the lipid/channel interaction with cloned Kir channels incorporated into a lipid bilayer, which allows us to manipulate the molecular structure of the lipids and the channels simultaneously. Using this system, they will further define the specific interaction between the lipid headgroup and the channel. They will explore the mechanism of this interaction by characterizing mutants specifically designed to evaluate the roles of the residues of interest. In addition, the same strategy will be used to study the effect of phosphatidic acid which initiates a special fast gating mode in Kir6.2 and Kir1.1 channels. They will test the second part of our hypothesis by changing the number, position, length and saturation of the lipid hydrocarbon chain as well as the side chains of the transmembrane helices. These investigators expect that this research will not only provide new information about the role of lipid/channel interaction in the gating of Kir channels, but also offer a comprehensive theory applicable to other ion channels and membrane proteins in general.